Peroxidase enzymes are widely distributed in nature and are produced by a wide variety of plant species, the chief commercial source being horseradish (Armoracia rusticana) and soyabean (Glycine max). The horseradish roots are harvested and the sprouted roots are crushed and mixed mechanically with water and a series of ammonium sulphate and ethanol precipitations are carried out for purification. High purity horseradish peroxidase enzyme (HRP) is obtained by conventional chromatographic techniques. But this extraction procedure from the general plant body leads to ecological problems by the dispersal of large quantities of waste and also there is a significant irreversible loss of enzyme activity because of ethanol and ammonium sulphate precipitation (Fowler et al, U.S. Pat. No. 5,728,550). Various other sources have been suggested in the literature for the production of peroxidase. For example, there is a report on the production of peroxidase from the bark of Hevea brasiliensis (Phytochemistry, 1997, vol 44, No. 2, pp 237-241); but exact specific activity is not shown. In another report, radish (Raphanus sativus) plant cell cultures have been suggested as a commercial source of extra-cellular peroxidase (Plant Cell, Tissue and Organ Culture, 1989, 18:321-327) but the yield and specific activity of the product enzyme are comparatively low.
Several references can be quoted for the use of calli as a source of peroxidase though many of them are of the opinion that callus culture is incapable of large-scale commercial production. Such suggestions are contained in Japanese Patent Applications numbered, JP-A-1222776 (source species: Trifolium repens L., Carica papaya L., Phellodendron amurense Rupr., Oenothera lamarchiana Ser., Scopolia japonica Maxim, Lithospermum erythrorhizon Sieb et Zucc., Glycine max Merrill, and Gynastemma pentaphyllum Makino; JP-A-63233782 (source species: general); JP-A-1222777 (source species: Zoysia japonica, and Zoysia macrostachchya); JP-A-62138188 (source species: Ipomoea aquatica Forsk); JP-A-1222778 (source species: Glycyrrhiza glabra L. var, Ipomoea batatas Lam. vat deulis Makino, Stevia rebaudiana Bettoni, and Bupleurum falcatum L.).
Reference may be made to Phytochemistry, 1998, Vol 49, pp 1219-1225 wherein there is a correlation between plant growth regulators and enzyme activity in cell suspension cultures of Catharanthus roseus. Correlation between extra-cellular peroxidase production and plant cell growth has been shown in Applied Biochemistry and Biotechnology, 1990, Vol 24/25, pp 213-222 using plant cell cultures of Artemesia annua, Coleus blumei, Pisum sativum, and Salvia officinalis. In both these cases, no indication is given for its use as a commercial source.
Reference may be made to the International Patent Application, Publication No. WO91/10729, which describes the production of plant cell suspension (root) cultures of Acer pseudoplatanus as a convenient source of peroxidase that can be easily recovered. Reference may be made to the U.S. Pat. No. 5,728,550, by Fowler et .al 1998, which reports the production of peroxidase from Acer pseudoplantanus callus dispersed in liquid culture containing confectionery waste. The drawback is that the enzyme production has not been optimized with growth regulators.
Reference may also be made to the U.S. Pat. No. 59-028473, U.S. Pat. No. 5,70,357, Stepan-Sarkissian et al. 1997, where extracellular peroxidase activity is seen in Theobroma. cacao and Coleus. blumei and intra-cellular enzyme in Santalum. alba. Reference may be made to the two publications titled “Extracellular peroxidases from cell suspension cultures of Vaccinium myritillus. Purification and characterization of two cationic enzymes” by Melo et al in Plant Sciences 106 (1995) 177-184 and in Plant Sciences 122 (1997) 1-10. The drawback is that the specific activity of the enzyme is very low, in the order of 75 U/mg only. Reference may be made to the publication in Plant physiology Biochemistry 39 (2001) 479-486 “Purification and stability of a basic peroxidase from strawberry callus culture”. The drawback is that the specific activity is extremely low, 0.56 n Katals/mg, and is not economically viable.
A method for purification of peroxidase by aqueous-organic phase separation is stated by Pokora et al (U.S. Pat. No. 4,992,372). No indication is given for further purification or the specific activity of the purified enzyme.